Group B streptococcus (GBS) is the most significant pathogen causing neonatal sepsis and meningitis. Of the 5 GBS serotypes, type III is responsible for the majority of GBS infections. Type III specific polysaccharide capsular antigen (TSA III) is one of the major virulence factors. This investigation seeks to further define the role of TSA III in GBS virulence by understanding the molecular genetics of capsulation. This proposal will extend the investigation started by the R23 grant which began to identify and characterize the organization, control, and function of the TSA III genes using contemporary genetic techniques. TSA III mutants in a more virulent GBS III strain (which produces a large amount of TSA III) will be derived by transposon mutagenesis to study how alterations in TSA III gene expression effect interaction of GBS with specific host defenses in vitro. Prior TSA III mutants were derived similarly in a less virulent (less TSA III) GBS III strain. Transposon insertion sites in low and high TSA III producing strains will be compared by Southern hybridization analysis to locate unidentified TSA III genes. Cloning of chromosomal DNA from mutagenized strains will isolate unidentified TSA III genes and their location in GBS chromosome will be mapped relative to known TSA III genes. The genetic structure and organization of TSA III genes will be determined by nucleotide sequence analysis. The amount of TSA III mRNA synthesized by isogenic GBS type III strains versus the cloned TSA III genes in E. coli will be analyzed by Northern blot analysis to begin defining the regulation of TSA III gene expression. The protein gene products from cloned TSA III genes will be analyzed by in vivo and in vitro gene expression assays. Proteins secreted through the cytoplasmic membrane will be identified and mapped using alkaline phosphatase: TSA III gene fusions created when transposon TnphoA inserts into a cloned TSA III gene encoding a secreted protein. Localization of secreted proteins on the cell surface will be performed by transmission electron microscopy by an indirect immunogold technique. A repetitive 1 Kb DNA sequence was observed only in the chromosome of virulent GBS strains but not in less virulent strains. The association of this 1 kb sequence with virulence in several stains will be confirmed by Southern analysis. Nucleotide sequencing and Northern analysis will be performed to define its structure and regulatory influence on TSA III biosynthesis. Knowledge of the molecular and genetic events controlling the synthesis, transport, and secretion of TSA III will provide a foundation for understanding its role in invasive neonatal GBS infections.